Severe Acute Respiratory Syndrome (SARS) is a recently emerged viral disease caused by a coronavirus (SARS-CoV). In contrast to the situation with other viral systems, there remain many unanswered questions regarding the process of SARS-CoV entry into host cells, especially with regard to membrane fusion events. While SARS-CoV has not undergone re-emergence since the initial outbreak, there remains a continued threat of further zoonotic outbreaks of SARS-CoV, or other animal coronaviruses. A detailed understanding of virus entry will be essential in our ability to respond to future outbreaks. We propose two specific aims: 1) To determine the role of a proteolytic cleavage site in the SARS-CoV S2 domain controlling membrane fusion. Our Preliminary Results indicate that a novel proteolytic cleavage site 793-KPTKR-797 within the S2 domain of the SARS coronavirus (SARS-CoV) S protein is instrumental in controlling viral fusion. Mutation of basic residues in this region leads to a loss of trypsin-mediated fusion and the introduction of a furin recognition site in this position allows fusion in the absence of ACE2 (the SARS-CoV receptor) suggesting an important role in host range. We have also shown that cleavage at R797 also primes membrane fusion via the lung-expressed serine proteases TMPRSS2 and HAT. We propose a comprehensive mutagenic and biochemical study of the S2 cleavage site (S2'), with the goal of understanding how selective cleavage of the SARS-CoV S protein by different proteases regulate S priming and fusion activation during virus entry. 2) To characterize the role of neutrophil elastase on SARS-CoV activation via cleavage within the S2 domain. It is also known that SARS-CoV entry can be activated by elastase, a mechanism of infection enhancement that is especially important in the context of the pronounced inflammatory response seen in the lungs of SARS-CoV-infected individuals. Our Preliminary Results indicate that neutrophil elastase also cleaves in the vicinity of the S2'cleavage site, at residue T795. We propose a comprehensive study of the elastase cleavage site at S2', with the goal of understanding how cleavage of the SARS-CoV S protein by elastase impacts virus entry and fusion. This project has a focus on the SARS-CoV because of its high priority status;however our work is likely to be directly applicable to other virus systems. Overall our studies will reveal critical features of SARS-CoV fusion activation and host range. Notably the presence of a cleavage site within S2 are very highly conserved across the Coronaviridae, and are likely to be universal features;with the fusion reaction primed by different proteases depending on the individual virus and circumstances. These studies will provide a model for both pathogenesis of SARS-CoV, as well as an understanding how coronaviruses might overcome species-specificity and emerge into new hosts. PUBLIC HEALTH RELEVANCE: Coronaviruses have recently received much attention as agents of infectious disease, due to the outbreak of severe acute respiratory syndrome (SARS) in the spring of 2003. While the SARS-coronavirus has not undergone re-emergence since the initial outbreak, there remains a continued threat of further outbreaks of SARS-CoV, or of other novel coronaviruses. The ability of the SARS-coronavirus to recognize and undergo membrane fusion with host cells is a major factor in its host range. Our studies are designed to elucidate the molecular details of SARS-coronavirus fusion and entry, which will serve as a model for both pathogenesis of SARS-CoV and the development of new antiviral drugs, as well as to help us understand how coronaviruses might overcome species-specificity and emerge into new hosts.